The aim of this study was to develop an effective selective/differential medium for culturing environmental strains of the Bacteroides fragilis group (BFG). This goal was achieved by modifying standard commercial Bacteroides Bile Esculin Agar (BBE Agar). Bacteroides Bile Esculin Agar was combined with substances that inhibit the growth of non-BFG bacteria, mostly Klebsiella pneumoniae and Fusobacterium mortiferum. The strains isolated from standard and modified BBE Agar were identified as BFG strains by PCR and 16S rRNA gene sequencing. The supplementation of standard BBE Agar with colistin (40 mg L-1), kanamycin (400 mg L-1) and vancomycin (7.5 mg L-1) increases the effectiveness of BFG bacteria isolation from <10% to 35%, and additional Gram staining improves the effectiveness of bacterial isolation five-fold relative to standard BBE Agar. The results of the present study also suggest that the presence of the bfr gene is not a reliable indicator for the identification of BFG strains.